The biological removal of inorganic nitrogenous compounds, such as ammonium (NH4+) and nitrate (NO3) from aquatic systems has long been a topic of interest for wastewater engineers and other water treatment professionals. These compounds contribute to eutrophication and are toxic to many aquatic organisms; therefore their presence in treated wastewater and in clean water systems, such as ponds, lakes, and reservoirs, is undesirable (Shannon et al, 2008). In the past, combinations of autotrophic nitrifying and denitrifying bacteria (which convert NH4+ to N2, with NO3− as an intermediate) were believed to be the only method for effecting such remediation. However, the discovery of novel metabolic pathways among several bacterial taxa during the latter part of the 20th century forced a reevaluation of this paradigm (Schmidt et al, 1987).
Nitrifying and denitrifying bacteria are an integral part of the planet's Nitrogen Cycle. Three main types of bacteria catalyze the conversions shown above. Ammonia oxidizing bacteria (AOBs) are aerobic chemolithoautotrophs belonging to the phylum Proteobacteria, which contains species such as Nitrosomonas, Nitrosococcus, and Nitrospira (Koops and Pommererening-Röser, 2001). These convert ammonia (NH4+) into hydroxylamine (NH2OH) through the action of ammonia monooxygenase (Equation 1). Hydroxylamine is then converted to nitrite (NO2−) by hydroxylamine oxidoreductase (Equation 2). Doubling time for these organisms ranges from 8-24 hours depending on nutrient availability (Hommes et al, 2003).NH3+O2+2H++2e−→NH2OH+H2O  (1)NH2OH+H2O→NO2−+5H++4e−  (2)
A second group of Proteobacteria, called nitrite oxidizing bacteria (NOBs), then converts nitrite into nitrate (Equation 3) with the enzyme nitrite oxidoreductase (Prosser, 1989). These are also aerobic chemolithoautotrophs, among the most common being members of the genus Nitrobacter. These organisms have a maximum doubling time of 20 hours (Tramper and Grootjen, 1986).NO2−+H2O→NO3−+2H++2e−  (3)
Nitrate is then converted into N2 in a process called denitrification (Equation 4), which was long believed to be limited to bacteria such as Thiosphaera, Paracoccus and Pseudomonas and to eukaryotes such as algae and fungi (Shapleigh, 2006). However, recent studies have found that members of the genus Bacillus (heterotrophic organisms of the phylum Firmicutes) can perform denitrification as well (Verbaendert, 2011). During denitrification, nitrate is substituted for oxygen as a terminal electron acceptor; therefore, because oxygen is an energetically preferable electron acceptor, denitrification generally occurs in anoxic environments. Nitrate is converted to nitrite before being ultimately converted to N2.2NO3−+10e−+12H+→N2+6H2O  (4)
The discovery of anaerobic ammonia oxidizers, collectively referred to as “anammox” bacteria, of the phylum Planctomycetes and belonging to genera such as Brocadia provided a new method for remediating inorganic nitrogenous compounds in wastewater (Strous et al, 1999). Organisms such as B. anammoxidans carry out denitrification of nitrite, using ammonia as an electron donor, with H2O and N2 as end products (Equation 5). Though their metabolism of ammonia was seen as quite novel, these bacteria are notoriously slow growing (doubling time approaches 11 days) and their anaerobic ammonia metabolism is completely, albeit reversibly, inhibited by oxygen at concentrations as low as 2 μM (Jetten et al, 2001).NH4++NO2−→N2+2H2O  (5)
Practical applications of these bacterial systems are numerous. In Partial Nitrification reactors (Hellinga et al, 1998), AOBs are utilized to convert ammonia into nitrite. Rather than allowing the nitrite to be converted to nitrate by NOBs (which must be inhibited in these systems through temperature and pH controls) the nitrite enriched wastewater is instead added to a denitrification reactor and converted to N2 by denitrifying bacteria. This allows the denitrifying bacteria to conserve energy, as they do not need to derive their NO2− from NO3−. The Partial Denitrification process can also be coupled with an anammox reactor in a process known as SHARON (single reactor system for high activity ammonium removal over nitrite), which allows the anammox Planctomycetes to utilize both NH4+ and NO2− to effect denitrification (Hellinga et al, 1998). Canon (completely autotrophic nitrogen removal over nitrite) reactors employ aerobic nitrifying bacteria from the phylum Proteobacteria for nitrification and anaerobic Planctomycetes for denitrification (Third et al, 2001). Aerobic AOBs oxidize NH4+ to NO2− while consuming oxygen, which creates an anoxic environment in which anammox bacteria can thrive. In addition to being hindered by the extended startup times of Planctomycetes, this system is prone to a buildup of NO2− in the presence of excess O2. Finally, NOx processes (Bock et al 1996) involve augmenting cultures of aerobic Proteobacteria such as Nitrosomonas with nitrogen oxides, which stimulates the bacteria to perform nitrification and denitrification concurrently (Bock et al, 1996).
Heterotrophic nitrification involves the conversion of NH4+ to NO2− by heterotrophic bacteria which, unlike the autotrophic Nitrosomonas, rely on organic compounds as a carbon and energy source (Schreiber, 2009). Though known to take place among some bacteria such as Thiosphaera pantotropha and some species in the genus Pseudomonas, rates of nitrification and denitrification were observed to be slower among heterotrophs (Schmidt et al, 2003). Therefore, autotrophs were viewed as superior organisms for remediating inorganic nitrogenous compounds in wastewater. However, Kim et al (2005) observed aerobic nitrification and denitrification among several strains of Bacillus (phylum Firmicutes) at higher rates than had been observed previously among heterotrophs. Nitrogen balance revealed that some ammonia nitrogen had been completely lost from the system, presumably as N2. This suggested a less complicated metabolic pathway among Bacillus than exists among Proteobacteria and Planctomycetes, as well as a potential alternative to the current nitrification and denitrification systems dominated by autotrophs.